The long-term objective of the present proposal is to develop a detailed understanding of the molecular basis for red cell membrane physiology as it relates to its mechanical function. The principal aim of this renewal application is to critically define the origins of red cell membrane material behavior at the molecular level by combining new functional information on individual proteins with techniques developed by the applicant for characterizing the biophysical behavior of the membrane. The applicant proposes to accomplish this by means of three aims. Specific Aim 1 is to define the molecular basis for red cell membrane structural integrity, with particular emphasis on the contributions of lateral linkages of skeletal proteins to membrane cohesion. He will approach this by exploring the hypothesis that weakening of such linkages will lead to membrane skeletal failure, the proposal focusing on alpha and beta spectrin and protein 4.1. Efforts will focus on a number of functional domains of these molecules defined by current structure/function understanding. Incorporation of selected recombinant peptides, antibodies and Fab fragments will be used to interfere with specific functional domains so that changes in mechanical stability can be quantitated by ektacytometry. In addition, the applicant will explore the role of phosphorylation of spectrin and protein 4.1 and the effects of calcium/calmodulin in regulating membrane cohesion. These experiments will take the form of affecting phosphorylation of specific proteins and monitoring effect on membrane stability. In addition, he will continue to characterize the mechanical properties of available pathologic red cells with defined mutations to gain further insights into the functional domains of cytoskeletal proteins. Specific Aim 2 is to define the molecular basis for membrane failure that involves separation of the lipid bilayer from the underlying skeleton. Using fluoresceinated Fab specific for certain proteins, he will first document the fluorescence density map of the labeled components using his new and novel technique of fluorescence-imaged micropipette aspiration, as well as use FRAP to identify which proteins are truly involved in linking the bilayer to the membrane. Having done so, he will use the above techniques to cause dissociation of integral membrane proteins from the skeleton to discern effect on membrane cohesion (assessed by mechanical stability). As part of these studies, contributions of ankyrin, 4.1, and p55 also will be examined. Likewise, available pathologic red cells will be used to examine effect of reconstitution on selective deficiencies. The data from this specific aim are expected to define the molecular basis for membrane failure. Specific Aim 3 will define a contribution of bilayer-skeletal protein network linkages to membrane deformability, following up the applicant's seminal observations regarding the effect of glycophorin A ligands on membrane rigidity. In particular, the effect that antibodies to glycophorin A have on band 3 mobility will be examined using various strategies of perturbation and reconstitution to selectively influence potential linkages. The third aim is expected to provide a molecular definition of the basis for regulation of membrane rigidity.